Articles
http://dx.doi.org/10.5012/bkcs.2013.34.2.367
Synthesis, Spectroscopic Studies of Binuclear Ruthenium(II) Carbonyl Thiosemicarba- zone Complexes Containing PPh
3/AsPh
3as Co-ligands: DNA Binding/Cleavage
K. Sampath, S. Sathiyaraj, and C. Jayabalakrishnan*
Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts and Science, Coimbatore – 641020, Tamil Nadu, India. *E-mail: drcjbstar@gmail.com
Received September 12, 2012, Accepted November 3, 2012
The ruthenium(II) ferrocenyl heterocyclic thiosemicarbazone complexes of the type [RuCl(CO)(EPh3)]2L (where E = P/As; L = binucleating monobasic tridendate thiosemicarbazone ligand) have been investigated.
Strutural features were determined by analytical and spectral techniques. Binding of these complexes with CT- DNA by absorption spectral study indicates that the ruthenium(II) complexes form adducts with DNA and has intrinsic binding constant in the range of 3.3 × 104- 1.2 × 105 M−1. The complexes exhibit a remarkable DNA cleavage activity with CT-DNA in the presence of hydrogen oxide and the cleavage activity depends on dosage.
Key Words : Binuclear ruthenium(II) complexes, Benzothiazolyl thiosemicarbazide, DNA interaction, Oxi- dative DNA cleavage
Introduction
Over a decade, the DNA-binding metal complexes have been extensively studied as DNA structural probes, DNA dependent electron transfer probes, DNA foot printing and sequence specific cleaving agents and potential anticancer drugs.1 Despite a considerable amount of reported materials, the knowledge of the nature of binding of these complexes to DNA and their binding geometries has remained relative- ly modest. Transition metal complexes can interact non- covalently with nucleic acids by intercalation, groove bind- ing or external electrostatic binding.2 There has been tre- mendous interest in studies related to the interaction of transition metal ions with nucleic acid because of their relevance in the development of new reagents in biotechno- logy and medicine.3 These studies are also important to understand the toxicity of drugs containing metal ions.4 In particular, ruthenium complexes are presently the objective of great attention in the field of medicinal chemistry with low systemic toxicity.5 Also, ruthenium complexes appear to penetrate reasonably well into the tumour cells and bind effectively to DNA.6 Moreover, the binuclear ruthenium complexes have been found to have better biological activity than mononuclear complexes.7
In the efforts to develop drugs with such capabilities, scientists have focused upon many different aspects of bio- logy during their research. Among the drugs discovered in recent years, various benzothiazoles8 possess potent cyto- toxicity properties. Thiazoles represent a very interesting class of compounds because of their wide applications in pharmaceutical, analytical and industrial aspects.9 In addi- tion, benzothiazolyl thiosemicarbazones and ferrocenyl thio-
semicarbazones were found to be active against tumours and fungi.10,11 The substitution of bulky group at the terminal nitrogen of the thiosemicarbazones considerably increases the biological activity.12 With this aid, the incorporation of binucleating thiosemicarbazone into ruthenium tertiary pho- sphine/ arsine complexes was initially aimed and promoted such compounds towards the DNA binding and DNA cleavage studies.
Experimental
Reagents and Methods. All the chemicals used were of AR grade. Solvents were purified and dried according to the standard procedure.13 RuCl3·3H2O was purchased from Loba Chemie and was used without further purification. Calf- thymus (CT-DNA) was purchased from Bangalore Genei, Bangalore, India. Micro analyses (C, H, N & S) were per- formed on a Vario EL III CHNS analyser at STIC, Cochin University of Science and Technology, Kerala, India. IR spectra were recorded as KBr pellets in the 400-4000 cm−1 region using Perkin Elmer FT-IR 8000 spectrophotometer.
Electronic spectra were recorded in dimethylsulphoxide solution with a Systronics double beam UV-vis spectrophoto- meter 2202 in the range 200-800 nm. 1H, 13C and 31P NMR spectra were recorded on a Bruker AV III 500 MHZ instru- ment using TMS and ortho phosphoric acid as an internal reference. EI mass spectrum of the complexes was recorded on a JEOL GCMATE II mass spectrometer. Melting points were recorded on a Veego VMP-DS model heating table and were uncorrected. Benzothiazolyl thiosemicarbazide14 and the metal precursors [RuHCl(CO)(PPh3)3]15 and [RuHCl- (CO)(AsPh3)3]16 were prepared according to the reported
procedures.
Synthesis of Thiosemicarbazone Ligands. To a meth- anolic solution (20 mL) of 6-nitro/H-2-benzothiazolyl thio- semicarbazide (20 mmol), diacetyl ferrocene (10 mmol) in methanol was added and stirred along with a few drops of glacial acetic acid. The mixture was then refluxed for about 8 h. The resultant product was washed with methanol and the purity of the ligands was checked by TLC and was further purified by column chromatography using petroleum ether and ethyl acetate mixture (60:40). The outline of syn- thesis of thiosemicarbazone ligands is given in Scheme 1.
H2L1: Yield 82%; Colour: Dark brown; mp 298 oC. Anal.
Calc for C30H24N10S4FeO4: C, 46.63; H, 3.13; N, 18.13; S, 16.60. Found: C, 46.62; H, 3.10; N, 18.23; S, 16.69. IR (cm−1):
1649 (C=N), 826 (C=S), 1511 (C=N thiazole ring). UV-vis (in DMSO); λmax, nm; 308, 368, 430. 1H NMR (ppm); 7.39- 8.23 (m, Ar-H), 2.28 (s, CH3), 8.67 (s, NH), 11.3 (s, SH), 4.59-4.79 (m, Cp ring-H). 13C NMR (ppm); 117-132 (Ar, C), 27 (CH3), 141 (C-S), 159 (C=N imine), 165 (thiazole C=N), 71-81 (C, Cp ring).
H2L2: Yield 79%; Colour: Black; mp 289 oC. Anal. Calc for C30H26N8S4Fe: C, 52.78; H, 3.84; N, 16.41; S, 18.79.
Found: C, 52.70; H, 3.82; N, 16.42; S, 18.89. IR (cm−1):
1631 (C=N), 807 (C=S), 1543 (C=N thiazole ring). UV-vis (in DMSO); λmax, nm; 305, 368, 428. 1H NMR (ppm); 7.41- 8.23 (m, Ar-H), 2.29 (s, CH3), 8.68 (s, NH), 11.9 (s, SH), 4.60-4.80 (m, Cp ring-H). 13C NMR (ppm); 119-131 (Ar, C), 27 (CH3), 140 (C-S), 157 (C=N imine), 165 (thiazole C=N), 71-81 (C, Cp ring).
Synthesis of Binuclear Ruthenium(II) Thiosemicarba- zone Complexes. All the reactions were carried out under anhydrous condition. The monobasic tridentate thiosemi- carbazones (0.1 mmol) were added to a solution of [RuHCl- (CO)(EPh3)3] (0.1 mmol), (E=P/As) in 1:2 molar ratio in
benzene-methanol/tetrahydrofuran (50 mL) mixture, and then refluxed for 10 h. The resulting solution was concent- rated to about 3 cm3 and the complexes were precipitated by the addition of small quantity of petroleum ether (60-80oC).
The complexes were then filtered off, washed with petro- leum ether and recrystallized from tetrahydrofuran/CH2Cl2/ petroleum ether and dried under vacuum. Schematic route for synthesis of binuclear ruthenium(II) thiosemicarbazone complexes is given in Scheme 2.
[RuCl(CO)(PPh3)]2L1: Yield 62%; Colour: Brown; mp
> 300 oC. Anal. Calc for C68H52N10Ru2FeO6P2S4Cl2: C, 50.28;
H, 3.23; N, 8.62; S, 7.90. Found: C, 50.18; H, 3.13; N, 8.61;
S, 7.93. EI-MS: Found m/z = 1624 (M+) (calculated m/z = 1624.30 for M+). IR (cm−1): 1603 (C=N), 745 (C–S), 1461 (C=N thiazole ring), 1952 (CO). UV-vis (in DMSO); λmax, nm; 305, 368, 430, 468. 1H NMR (ppm); 7.20-8.25 (m, Ar- H), 2.34 (s, CH3), 8.67 (s, NH), 4.55-4.78 (m, Cp ring-H).
13C NMR (ppm); 116-134 (Ar, C), 27 (CH3), 136 (C-S), 179 (C=N), 201 (CO), 69-79 (C, Cp ring). 31P NMR (ppm); 42 (P, PPh3).
[RuCl(CO)(AsPh3)]2L1: Yield 59%; Colour: Black; mp
> 300 oC. Anal. Calc for C68H52N10Ru2FeO6As2S4Cl2: C, 47.70; H, 3.06; N, 8.18; S, 7.49. Found: C, 47.81; H, 3.26;
N, 8.28; S, 7.39. EI-MS: Found m/z = 1712 (M+) (calculated m/z = 1712.20 for M+). IR (cm−1): 1619 (C=N), 737 (C–S), 1469 (C=N thiazole ring), 1954 (CO). UV-vis (in DMSO);
λmax, nm; 308, 368, 432, 471. 1H NMR (ppm); 7.28-8.24 (m, Ar-H), 2.35 (s, CH3), 8.69 (s, NH), 4.56-4.78 (m, Cp ring- H). 13C NMR (ppm); 117-133 (Ar, C), 27 (CH3), 136 (C-S), 178 (C=N), 201 (CO), 69-79 (C, Cp ring).
[RuCl(CO)(PPh3)]2L2: Yield 58%; Colour: Black; mp
> 300 oC. Anal. Calc for C68H54N8Ru2FeO2P2S4Cl2: C, 53.23; H, 3.55; N, 7.30; S, 8.36. Found: C, 53.21; H, 3.65;
N, 7.32; S, 8.37. EI-MS: Found m/z = 1534 (M+) (calculated Scheme 1. Synthesis of binucleating thiosemicarbazone ligands. Scheme 2. Structure of binuclear ruthenium(II) thiosemicarbazone
complexes.
m/z = 1534.31 for M+). IR (cm−1): 1609 (C=N), 748 (C–S), 1461 (C=N thiazole ring), 1963 (CO). UV-vis (in DMSO);
λmax, nm; 305, 368, 430, 464. 1H NMR (ppm); 7.13-8.24 (m, Ar-H), 2.30 (s, CH3), 8.68 (s, NH), 4.61-4.81 (m, Cp ring- H). 13C NMR (ppm); 117-135 (Ar, C), 30 (CH3), 137 (C-S), 172 (C=N), 198 (CO), 69-73 (C, Cp ring). 31P NMR (ppm);
42 (P, PPh3).
[RuCl(CO)(AsPh3)]2L2: Yield 61%; Colour: Brown; mp
> 300 oC. Anal. Calc for C68H54N8Ru2FeO2As2S4Cl2: C, 53.23;
H, 3.55; N, 7.30; S, 8.36. Found: C, 53.21; H, 3.65; N, 7.32;
S, 8.37. EI-MS: Found m/z = 1622 (M+) (calculated m/z = 1622.20 for M+). IR (cm−1): 1613 (C=N), 738 (C–S), 1461 (C=N thiazole ring), 1963 (CO). UV-vis (in DMSO); λmax, nm; 305, 365, 431, 469. 1H NMR (ppm); 7.27-8.24 (m, Ar- H), 2.08 (s, CH3), 8.69 (s, NH), 4.60-4.85 (m, Cp ring-H).
13C NMR (ppm); 118-133 (Ar, C), 31 (CH3), 136 (C-S), 172 (C=N), 199 (CO), 68-73 (C, Cp ring).
DNA-Binding and Cleavage Assay.
Electronic Absorption Spectroscopy: All the experiments involving the binding of binuclear ruthenium(II) complexes with CT-DNA were carried out in a doubly distilled water buffer with tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid. A solution of CT-DNA in the buffer gave a ratio of UV absorbance of about 1.8-1.9 at 260 and 280 nm, indicating that the DNA was sufficiently free of pro- tein.17 The CT-DNA concentration per nucleotide was deter- mined spectrophotometrically by employing an extinction coefficient of 6600 M−1 cm−1 at 260 nm. The complexes were dissolved in a mixed solvent of 5% DMSO and 95%
Tris-HCl buffer. Stock solutions were stored at 4 °C and used within 4 days. Absorption titration experiments were performed with fixed concentration of the complexes (25 µM) with varying concentration of DNA (0-40 µM). While measuring the absorption spectra, an equal amount of DNA was added to both the test solution and the reference solution to eliminate the absorbance of DNA itself.
DNA Cleavage: The DNA cleavage activity of the ligands and ruthenium(II) complexes were monitored by agarose gel electrophoresis on CT-DNA. The tests were performed under aerobic condition with H2O2 as an oxidant. Each reaction mixture contained 30 µM of CT-DNA, 30 and 60 µM of each compound in DMSO and 60 µM of hydrogen peroxide in 50 mM Tris-HCl, (pH 7.2). The reaction was incubated at 37 oC for 2 h. After incubation, 1 µL of loading buffer (0.25% bromophenol blue, 0.25% xylene cynol and 60%
glycerol) was added to the reaction mixture and loaded onto a 1% agarose gel containing 1.0 µg/mL of ethidium bro- mide. The electrophoresis was carried out for 2 h at 50 V in Tris-acetic acid-EDTA buffer. The bands were visualized under UV light and photographed.
Results and Discussion
Structure of the Complexes. A new series of binuclear ruthenium(II) thiosemicarbazone complexes of the type [RuCl(CO)(EPh3)]2L (E=P/As; L = binucleating monobasic
tridentate thiosemicarbazone ligands) were synthesized by reacting ruthenium(II) precursors [RuHCl(CO)(EPh3)3] with binucleating monobasic tridentate thiosemicarbazone ligands in 2:1 molar ratio, respectively in benzene-methanol/tetra- hydrofuran mixture. The synthesized binuclear ruthenium(II) thiosemicarbazone complexes are stable in air at room temperature, non-hygroscopic in nature and soluble in com- mon solvents such as dichloromethane, dimethylformamide, dimethylsulphoxide, etc. The analytical data of the com- plexes are in good agreement with the calculated values thus confirming the proposed hetero bimetallic composition for all the complexes.
IR Spectra. The important IR spectral data of the ligands were compared with those of the binuclear ruthenium(II) complexes in order to confirm the binding mode of the thio- semicarbazone ligands to ruthenium atom in the complexes.
In principle, the thiosemicarbazone ligands exhibit thione - thiol tautomerism, since it contains a thioamide –NH-C=S functional group.18 The free ligands display ν(C=S) absorptions in the region 807-826 cm−1, shifts to 737-748 cm−1 in the spectra of the complexes. This observation may be attributed to the enolization of –NH-C=S and subsequent coordination through the deprotonated sulphur.19 A strong band in the range 1631-1649 cm−1 due to the azomethine group (C=N) of free ligands was shifted to lower frequency in the spectra of complexes in the range 1603-1619 cm−1 indicating that the other coordination is through azomethine nitrogen atom.20 IR spectrum of the ligands revealed a medium intensity band in the region 1511-1543 cm−1 ν(C=N) thiazole ring, which is shifted to lower frequency in the range 1461-1469 cm−1 after complexation, which also indicates that it has been affected upon coordination to the metal ion.21 The ν(C-S-C) at 621 cm−1 of the thiazole ring remains unchanged which demonstrated that the thiazole group of sulphur does not coordinate to the ruthenium metal.22 For all complexes, a strong band in the region 1952-1963 cm−1 is due to terminally coordinated carbonyl group and is observed at higher frequency than in the precursor complexes.18 Absorptions at 513-521 cm−1 in the spectra of the complexes are attributed to the ν(Ru-N).19 In addition, other characteristic bands due to triphenylphos- phine/triphenylarsine are also present in the expected region.18 Electronic Spectra. The absorption of free ligands show- ed two types of transitions appeared in the range 305-308 nm and at 368 nm are due to π-π* and n-π* transitions involving molecular orbital of the cyclopentadienyl ring, C=N and enolic -SH chromophore respectively. For all the ligands and complexes, the shoulder in the region 428-430 nm is attributed to the transition of 3d electron on iron to either non-bonding or antibonding orbitals of the cyclo- pentadienyl ring.11 These bands are shifted in the spectra of the complexes, indicating the involvement of imine group nitrogen and thionyl sulphur in coordination with central metal atom. The spectra of the complexes showed five bands in the region 305-471 nm. The ground state of ruthenium(II) in an octahedral environment is 1A1g, arising from the t2g6
configuration, and the excited states corresponding to the t2g5eg1 configuration are 3T1g, 3T2g, 1T1g and 1T2g. Hence, four
bands corresponding to the transitions 1A1g → 3T1g, 1A1g →
3T2g, 1A1g → 1T1g and 1A1g → 1T2g are possible in order of the increasing energy. The lower wavelength bands are charac- terized as ligand centered transitions occurring within the ligand orbitals. The bands appearing in the region 464-471 nm have been assigned to charge transfer transitions arising from excitation of an electron from the metal t2g level to an unfilled molecular orbital derived from the π* level of the ligands.23 The pattern of the electronic spectra for the com- plexes indicated the presence of an octahedral environment around ruthenium(II) atom similar to that of the other ruthenium(II) octahedral complexes.11,23
1H, 13C and 31P NMR Spectra. The formation of ligands and complexes were conveniently monitored by peak ratios in the 1H, 13C and 31P NMR spectra. The 1H NMR spectra of H2L2, {[RuCl(CO)(PPh3)]2L2} and 31P NMR spectrum of {[RuCl(CO)(PPh3)]2L2} are given in Figure S1-S3 respec- tively. The signal due to the -SH proton in the ligands at 11.3-11.9 ppm disappears in the case of metal complexes, showing the bonding of thiolic sulphur to the metal after the deprotonation of the functional group.24 The signal observed at 8.67-8.69 ppm is due to -NH proton for free ligands and the complexes.25 For ligands and complexes, the chemical
shift at 4.55-4.85 ppm is due to cyclopentadienyl ring pro- tons.26 Of the slight chemical shifts were observed for ferro- cene in the complexes when compared to ligands, reflect extension of the delocalized π-system of the thiosemi- carbazide chain into cyclopentadienyl ring.27 The ligands and complexes shows signal at 2.08-2.35 ppm due to methyl group.
13C NMR spectra of all the ligands displayed a single re- sonance at 157-159 ppm due to the azomethine carbon atoms, which also confirms the structure of the ligands.28 The signal at 165 ppm corresponds to thiazolic C=N carbon.29 The downfield shift of these two signals at 172-179 ppm clearly indicates that both the C=N carbons are affected by coordination.30 The thiolic carbon of the thiosemicarbazones appeared at 140-141 ppm. Upon coordination, this signal was shifted to upfield and appeared at 136-137 ppm.24 The signal due to methyl carbon of the ligands and complexes appears at 27-31 ppm. The aromatic carbons of free ligands and the complexes show signal in the region 116-135 ppm.
For ligands and complexes, the signal at 68-81 ppm is assign- ed to cyclopentadienyl ring carbons.31 For all the complexes, the terminal carbonyl group, CO appeared in the range 198- 201 ppm.32
Figure 1. EI-Mass spectra of the complexes, {[RuCl(CO)(PPh3)]2L1}, {[RuCl(CO)(AsPh3)]2L1}, {[RuCl(CO)(PPh3)]2L2} and {[RuCl(CO)(AsPh3)]2L2}.
31P NMR spectrum of the complexes [RuCl(CO)(PPh3)L1] and [RuCl(CO)(PPh3) L2] were recorded in order to confirm the presence of triphenylphosphine group. The observation of a sharp singlet at 42 ppm confirms the presence of only one triphenylphosphine group in the complexes.
Mass Spectral Analysis. The mass spectra of the binuclear ruthenium(II) complexes were in good agreement with the proposed structure and are shown in Figure 1. The molecular ion peak, M+ appeared at m/z = 1624, 1712, 1534 and 1622 confirms the stoichiometry of the complexes, {[RuCl(CO)- (PPh3)]2L1}, {[RuCl(CO)(AsPh3)]2L1}, {[RuCl(CO)(PPh3)]2L2} and {[RuCl(CO)(AsPh3)]2L2}, respectively.
DNA-Binding and Cleavage Assay.
Electronic Absorption Spectroscopy: Electronic absorp- tion spectroscopy is one of the most common techniques for the investigation of the mode of interaction of metal com- plexes.33,34 Absorption spectra of the complexes in the absence and presence of CT-DNA is given in Figure 2. The binding of the complexes has been characterized classically through absorbance and shift in the wavelength as a function of added concentration of DNA. Upon addition of increasing amount of CT-DNA, a significant hyperchromic effect in the intraligand bands at 257-436 nm was observed accompanied by a moderate hypsochromism of 2-3 nm, indicative of stabilizing of the DNA helix. The metal complexes which non-intercalatively or electrostatically bind with DNA may result in hyperchromism and hypsochromism.35,36 The absorp- tion spectra show clearly that the addition of DNA to the complexes leads to strong hyperchromism accompanied by slight hypsochromism to the [DNA]/[Ru]. Observed spectral
characteristics suggest that all the complexes interact with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA via electrostatically. In order to compare quantitatively the binding strength of the compounds, the intrinsic binding constants (Kb) of them with CT-DNA were determined from the following equation,37
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb (εb – εf) where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient εa, εf and εb correspond to Aobs/[compound], the extinction coefficient of the free compound and the extinction coefficient of the compound when fully bound to DNA, respectively. The plot of [DNA]/
(εa – εf) versus [DNA] gave a slope and the intercept which are equal to 1/(εb – εf) and 1/Kb (εb – εf), respectively; Kb is the ratio of the slope to the intercept. The magnitudes of intrinsic binding constant, Kb of the binuclear ruthenium(II) complexes has been estimated to be of 1.2 × 105 M−1 {[RuCl(CO)(PPh3)]2L1}, 1.0 × 105 M−1 {[RuCl(CO)(AsPh3)]2L1}, 3.6 × 104 M−1 {[RuCl(CO)(PPh3)]2L2}, 3.3 × 104 M−1 {[RuCl- (CO)(AsPh3)]2L2} respectively. This is good agreement with the reported literature.38 From the results obtained, it has been found that the complex, {[RuCl(CO)(PPh3)]2L1} strong- ly bound with CT-DNA than that of other complexes.
DNA Cleavage Activity. After binding to DNA, duly designed metal complexes can induce several changes in DNA conformation. Metal complexes, which could induce DNA deformation, such as bending, ‘local denaturation’
(overwinding and underwinding), intercalation, micro loop Figure 2. Absorption spectral traces of the complexes, {[RuCl(CO)(PPh3)]2L1}, {[RuCl(CO)(AsPh3)]2L1}, {[RuCl(CO)(PPh3)]2L2} and {[RuCl(CO)(AsPh3)]2L2} with CT-DNA.
formation and subsequent DNA shorting lead to decrease in molecular weight of DNA. Gel electrophoresis is an exten- sively used technique to study interaction of compounds with DNA; in this method segregation of the molecules will be on the basis of their relative rate of movement through a gel under the influence of electric field.39
The ability of metal complexes to perform DNA cleavage is generally monitored by agarose gel electrophoresis and in the present work CT DNA was chosen to investigate its cleavage. The cleavage experiments were carried out in the absence and presence of activating agent, H2O2 under aero- bic condition. The hydrolytic cleavage activity of the bi- nuclear ruthenium(II) complexes does not show any signi- ficant cleavage of DNA (not shown) when applied separate- ly. This result suggests that the nuclease activity of the complexes does not involve by hydrolytic pathway. Further,
the ferrocenyl thiosemicarbazone ligands were subjected to nuclease activity in the presence of H2O2 and are shown in Figure 3. A control experiment using DNA with H2O2 does not show any significant cleavage of DNA (Lane 1). Upon electrophoresis, there is no considerable difference in the intensity of the bands observed for the ligands bound DNA as compared to control DNA. This reveals that the ferro- cenyl thiosemicarbazone ligands are not active to cleave DNA.
These experimental facts demonstrate that, the binuclear ruthenium(II) thiosemicarbazone complexes with an activat- ing agent, H2O2 are required to show effective cleavage of CT DNA. Figure 4 shows that the oxidative cleavage of CT DNA induced by binuclear ruthenium(II) complexes in the presence of H2O2. Control experiment using DNA alone (Lane 1) does not show any significant cleavage of CT-DNA even on longer exposure time. When the DNA is allowed to interact with the complexes at various concentrations (30 and 60 µM), a substantial decrease in the intensities of the bands for the metal bound DNA as compared to untreated control DNA was observed, which suggests the cleavage of DNA by ruthenium(II) complexes.40 When the concentration was increased to 60 μM for all the complexes, the bands (Lane 3, 5, 7 & 9) were completely disappeared, indicating the sufficient cleavage of DNA. The results showed that {[RuCl(CO)(PPh3)]2L1} has more cleavage activity than the other complexes and the efficiency of the complexes are comparable to those of other hetero bimetallic ruthenium complexes.41,42 This phenomenon indicates that the com- plexes with H2O2 are capable to cleave the DNA; as a con- sequence, these complexes are better suited for therapeutic applications particularly, in cancer chemotherapeutics.43
Conclusion
The binuclear ruthenium(II) complexes of benzothiazole substituted ferrocenyl thiosemicarbazone were synthesized and characterized by various physico-chemical and spectro- scopic methods. The complexes are tentatively assigned for an octahedral geometry. The DNA binding study suggests that all the complexes interact with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA via electro- statically. The magnitudes of intrinsic binding constant, Kb
of the binuclear ruthenium(II) complexes has been estimated in the range 3.3 × 104 M−1- 1.2 × 105 M−1. The binding affinity of the complexes were in the order {[RuCl(CO)- (PPh3)]2L1} > {[RuCl(CO)(AsPh3)]2L1} > {[RuCl(CO)(PPh3)]2L2}
> {[RuCl(CO)(AsPh3)]2L2}. DNA cleavage studies reveal that the complexes have the ability to cleave nucleic acids and the extent of the cleavage was found to be dose dependent. The complex, {[RuCl(CO)(PPh3)]2L1} has more cleavage activity than the other complexes.
Acknowledgments. We sincerely thank Council of Scientific and Industrial Research (CSIR), New Delhi, for financial support [Scheme. No. 01(2308)/09/EMR-II].
Figure 3. Gel electrophoresis diagram showing the cleavage of CT-DNA (30 µM) incubated at 37 °C for a period of 2 h at a concentration of 30 and 60 µM of ligands in 5% DMSO and 95% 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2. Lane 1: DNA+ 60 µM H2O2; Lanes 2 and 3: DNA + 60 µM H2O2 + 30 and 60 µM of H2L1, respectively; Lanes 4 and 5: DNA + 60 µM H2O2 + 30 and 60 µM of H2L2, respectively.
Figure 4. The gel electrophoresis showing the chemical nuclease activity of the CT-DNA (30 µM) with different concentration of the complexes; Lane 1: DNA control; Lane 2: 30 µM {[RuCl(CO)(PPh3)]2L1} + DNA + 60 µM H2O2; Lane 3: 60 µM {[RuCl(CO)(PPh3)]2L1} + DNA + 60 µM H2O2; Lane 4: 30 µM {[RuCl(CO)(AsPh3)]2L1} + DNA + 60 µM H2O2; Lane 5: 60 µM {[RuCl(CO)(AsPh3)]2L1} + DNA + 60 µM H2O2; Lane 6: 30 µM {[RuCl(CO)(PPh3)]2L2} + DNA + 60 µM H2O2; Lane 7: 60 µM {[RuCl(CO)(PPh3)]2L2} + DNA + 60 µM H2O2; Lane 8: 30 µM {[RuCl(CO)(AsPh3)]2L2} + DNA + 60 µM H2O2; Lane 9: 60 µM {[RuCl(CO)(AsPh3)]2L2} + DNA + 60 µM H2O2.
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